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Not applicable.
Despite advances in medical science and new drugs, malaria, filariasis, dengue and the viral encephalilitides remain important diseases of humans, with an estimated 2 billion people worldwide living in areas where these are endemic (The World Health Reportxe2x80x941999, World Health Organization, Geneva, Switzerland (1999)). The causative agents of these diseases are transmitted by mosquitoes, and therefore disease control methods have relied heavily on broad spectrum chemical insecticides to reduce mosquito populations. However, chemical insecticide usage is being phased out in many countries due the development of insecticide resistance in mosquito populations. Furthermore, many governments restrict use of these chemicals because of concerns over their effects on the environment, especially on non-target beneficial insects, and vertebrates through contamination of food and water supplies.
As a result of these problems, the World Health Organization is facilitating the replacement of chemical with bacterial-based insecticides through the development of standards for their registration and use (Guideline specifications for bacterial larvicides for public health use, WHO Document WHO/CDS/CPC/WHOPES/99.2, World Health Organization, Geneva, Switzerland (1999)). Products based on bacteria have been designed to control mosquito larvae, and the two most widely used are Vectobac(copyright) and Teknar(copyright), both of which are based on Bacillus thuringiensis subsp. israelensis. In addition, Vectolex(copyright), a new product based on B. sphaericus has come to market recently for control of the mosquito vectors of filariasis and viral diseases. These products have achieved moderate commercial success, but their high cost and lower efficacy compared to many chemical pesticides prevents them from being used more extensively in many developing countries. Moreover, concerns have been raised about their long term utility due to resistance, which has already been reported to B. sphaericus in field populations of Culex mosquitoes in India, Brazil, and France (Sinxc3xa8gre, et al. First field occurrence of Culex pipiens resistance to Bacillus sphaericus in southern France, VII European Meeting, Society for Vector Ecology, 5-8 September Barcelona, Spain (1994); Rao et al., J. Am. Mosq. Control Assoc. 11:1-5 (1995); Silva-Filha et al., J. Econ. Entomot 88: 525-530 (1995)).
The insecticidal properties of these bacteria are due primarily to insecticidal proteins produced during sporulation. In Bacillus thuringiensis subsp. israelensis (Bti), the key proteins are Cyt1A (27 kDa), Cry11A (72 kDa), Cry4A (128 kDa) and Cry4B (134 kDa), whereas B. sphaericus (Bs) produces 41- and 52-kDa proteins that serve, respectively, as the toxin and binding domains of a single binary toxin (Federici et al. in Bacterial Control of Mosquitoes and Blackflies, eds.: de Barjac and Sutherland, D. J, 11-44 (Rutgers University Press, New Brunswick, N.J.) (1990); Baumann et al., Microbiol. Rev. 55:425-436 (1991)).
Biochemical and toxicological differences in the Bti and Bs toxins suggested that it might be possible to construct an improved bacterium by combining their toxins into a single bacterium. Numerous attempts using this approach have been made over the past decade to create a recombinant bacterium with the desired toxicity. Several groups, for example, have introduced Bti toxin genes in Bs. For example, Bar et al., J. Invertebrate Pathol. 149-158 (1991 cloned Bti endotoxin genes into Bs 2362, but found that the biological activity of the recombinant organism was lower than that of Bti. Poncet et al., FEMS Microbiol Lett. 117:91-96 (1994) cloned the cry4B and cry11A genes of Bti into Bs 2297, and Poncet et al., Appl Environ, Microbiol. 63:4413-4420 (1997) introduced the cry11A gene into the same strain by homologous recombination. Thoxc3xa9ry et al., Appl. Environ. Microbiol. 64:3910-3916 (1998) introduced a Bt cyt1Ab1 gene into Bs, but reported that the level of expression of the cyt1Ab1 gene was probable too low to have any significant effect on toxicity. Servant et al., Appl Environ Microbiol. 65:3021-3026 (1999) introduced Cry11A and Cry11Ba Bt toxins in Bs 2297 by in vivo recombination, and showed that the host range could thereby be increased. Bourgouin et al., Appl. Environ Microbiol. 56:340-344 (1990) introduced Bs toxin into Bti, but found no synergistic or additive effect between the toxins against mosquito larvae. Attempts to combine the advantages of Bs and Bt in other manners have also apparently not proven commercially useful. Simply growing cultures of Bs and Bt and then combining the two organisms, for example, is not effective because the spores of the two organisms are considered to form too large a proportion of the resulting mix in proportion to the weight of the toxins to provide adequate toxicity.
Commercial development of new biopesticides is costly, in part because of EPA regulations requiring extensive testing, and margins are low relative to, for example, pharmaceutical agents. It does not appear that any of the recombinant organisms reported in the past decade have shown sufficient improvement over current commercial Bti or Bs strains to warrant development for commercial use in mosquito control.
The invention provides nucleotide sequences, expression vectors, host cells and methods for achieving the high level expression of Bs binary toxin, particularly in cells of Bacillus species and especially in Bs and in Bt cells.
In particular, the invention provides nucleic acid sequences comprising, in the following order, A nucleic acid sequence comprising, in the following order, a B. thuringiensis promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a B. sphaericus binary toxin. In some embodiments, the bacterial STAB-SD sequence is selected from the group consisting of GAAAGGAGG (SEQ ID NO:1), GAAGGGGGG (SEQ ID NO:2), GAGGGGGGG (SEQ ID NO:3), GAAAGGGGG (SEQ ID NO:4), GAAAGGAGG (SEQ ID NO:5), and GAAAGGGGT (SEQ ID NO:6). The B. thuringiensis promoter is a cry promoter, and in particular can be a cry1 promoter.
Further, the B. thuringiensis promoter can be cry1Aa1, cry1Aa2, cry1Aa3, cry1Aa4, cry1Aa5, cry1Aa6, cry1Ba1, cry1Ba2, cry1Ca1, cry1Ca2, cry1Ca3, cry1Ca4, cry1Ca5, cry1Ca6, cry1Ca7 cry1Fa1, cry1Fa2, cyt1Aa1, cyt1Aa2, cyt1Aa3, or cyt1Aa4. In some preferred embodiments, the B. thuringiensis promoter is a cyt1Aa1 promoter. The nucleic acid can have both a BtI promoter and a BtII promoter, and the two promoters can be overlapping.
The invention further provides expression vectors comprising the nucleic acid described above, and host cells comprising the expression vectors. The host cells can further comprise a 20 kD protein encoded by the Bti cry11A operon. In preferred embodiments, the host cell is a B. thuringiensis cell or a B. sphaericus cell.
The invention further provides a nucleic acid sequence comprising, in the following order, a B. thuringiensis promoter which binds a sigma factor A protein, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a B. sphaericus binary toxin.
The invention also relates to a method of enhancing production of B. sphaericus binary toxin in a host bacterial cell, said method comprising: transforming the host cell with a gene comprising, in the following order, a B. thuringiensis promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a B. sphaericus binary toxin; and expressing said gene in the host cell; whereby expression of said gene enhances production of B. sphaericus binary toxin as compared to production of B. sphaericus binary toxin in a wild-type B. sphaericus cell that is not transformed with said gene. The bacterial STAB-SD sequence used in the method can be selected from the group consisting of GAAAGGAGG (SEQ ID NO:1), GAAGGGGGG (SEQ ID NO:2), GAGGGGGGG (SEQ ID NO:3), GAAAGGGGG (SEQ ID NO:4), GAAAGGAGG (SEQ ID NO:5), and GAAAGGGGT (SEQ ID NO:6). The host cell used in the method can be a B. thuringiensis cell or a B. sphaericus cell. The host cell may further express a 20 kD product of a cry11A gene.
In other embodiments, the invention relates to a method of creating a recombinant bacterium, said method comprising the steps of: transforming the recombinant bacterium with a gene comprising, in the following order: a B. thuringiensis promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a B. sphaericus binary toxin; and expressing said gene in the host cell; whereby expression of said gene enhances production of B. sphaericus binary toxin as compared to production of B. sphaericus binary toxin in a wild-type B. sphaericus cell that is not transformed with said gene. The bacterial STAB-SD sequence used in the method can be selected from the group consisting of GAAAGGAGG (SEQ ID NO:1), GAAGGGGGG (SEQ ID NO:2), GAGGGGGGG (SEQ ID NO:3), GAAAGGGGG (SEQ ID NO:4), GAAAGGAGG (SEQ ID NO:5), and GAAAGGGGT (SEQ ID NO:6). The recombinant bacterium used in the method can be selected from the group consisting of B. thuringiensis, B. sphaericus, and a member of a Bacillus species other than Bti or Bs. 
The invention also relates to a method of increasing toxicity of a B. thuringiensis bacterium to a mosquito, said method comprising the steps of: transforming said bacterium with a nucleic acid sequence comprising, in the following order, a B. thuringiensis promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins a B. sphaericus binary toxin; and expressing said gene in the bacterium; whereby expression of said gene enhances production of B. sphaericus binary toxin as compared to production of B. sphaericus binary toxin in a wild-type B. sphaericus cell that is not transformed with said gene. The bacterium can further comprise a 20 kD product of the cry11A gene.
In another group of embodiments, the invention provides a recombinant cell of B. sphaericus, said cell comprising nucleic acid sequence comprising, in the following order, a B. thuringiensis promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a B. sphaericus binary toxin. The bacterial STAB-SD sequence present in the recombinant cell can be selected from the group consisting of GAAAGGAGG (SEQ ID NO:1), GAAGGGGGG (SEQ ID NO:2), GAGGGGGGG (SEQ ID NO:3), GAAAGGGGG (SEQ ID NO:4), GAAAGGAGG (SEQ ID NO:5), and GAAAGGGGT (SEQ ID NO:6). The B. thuringiensis promoter can be a cry promoter, or can be selected from the group consisting of cry1Aa1, cry1Aa2, cry1Aa3, cry1Aa4, cry1Aa5, cry1Aa6, cry1Ba1, cry1Ba2, cry1Ca1, cry1Ca2, cry1Ca3, cry1Ca4, cry1Ca5, cry1Ca6, cry1Ca7cry1Fa1, cry1Fa2, cyt1Aa1, cyt1Aa2, cyt1Aa3, and cyt1Aa4. In a preferred embodiment, the B. thuringiensis promoter is a cyt1Aa1 promoter. The recombinant cell can further express a 20 kD product of a cry11A operon.
In yet another set of embodiments, the invention provides a method for increasing toxicity of a B. sphaericus cell, said method comprising transforming the cell with a nucleic acid sequence comprising, in the following order, a B. thuringiensis promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a B. sphaericus binary toxin; and expressing said nucleic acid sequence in the host cell; whereby expression of said nucleic acid sequence enhances production of B. sphaericus binary toxin as compared to production of B. sphaericus binary toxin in a wild-type B. sphaericus cell that is not transformed with said nucleic acid sequence. The bacterial STAB-SD sequence can be selected from the group consisting of GAAAGGAGG (SEQ ID NO: 1), GAAGGGGGG (SEQ ID NO:2), GAGGGGGGG (SEQ ID NO:3), GAAAGGGGG (SEQ ID NO:4), GAAAGGAGG (SEQ ID NO:5), and GAAAGGGGT (SEQ ID NO:6). The B. thuringiensis promoter can be a cry promoter, or can be selected from the group consisting of cry1Aa1, cry1Aa2, cry1Aa3, cry1Aa4, cry1Aa5, cry1Aa6, cry1Ba1, cry1Ba2, cry1Ca1, cry1Ca2, cry1Ca3, cry1Ca4, cry1Ca5, cry1Ca6, cry1Ca7 cry1Fa1, cry1Fa2, cyt1Aa1, cyt1Aa2, cyt1Aa3, and cyt1Aa4. In a preferred embodiment, the B. thuringiensis promoter is a cyt1Aa1 promoter.
The invention also provides a method for suppressing resistance to a B. sphaericus binary toxin, said method comprising expressing a Bti Cyt1Aa1 protein in a B. sphaericus cell expressing said binary toxin, as well as a method for suppressing resistance to a B. sphaericus binary toxin, the method comprising expressing a Bti Cyt1Aa1 protein in a B. thuringiensis cell expressing said binary toxin. In yet another embodiment, the invention provides a method for suppressing resistance to a B. sphaericus binary toxin, the method comprising administering Bti Cyt1Aa1 protein with said binary toxin. The Bti Cyt1Aa1 protein can be in a powder of lysed, lyophilized Bti cells, or can be in the form of a purified protein. The Bti Cyt1Aa1 protein is administered in a Cyt1Aa1 protein to Bs ratio selected from about 1:2 to about 1:50. In especially preferred embodiments, the Bti Cyt1Aa1 protein is administered in a Cyt1Aa1 protein to Bs ratio of about 1:10.